Enhanced Herbicide Metabolism Research Articles

PfGSTF2 endows resistance to quizalofop-p-ethyl in Polypogon fugax by GSH conjugation.

Populations of Polypogon fugax have developed resistance to many acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. This resistance threats the effectiveness and sustainability of herbicide use. In our previous research, a field P. fugax population exhibited GST-based metabolic resistance to the widely used ACCase-inhibiting herbicide quizalofop-p-ethyl. Here, in this current study, we identified and characterized two GST genes (named as PfGSTF2 and PfGSTF58) that showed higher expression levels in the resistant than the susceptible population. Transgenic rice calli overexpressing PfGSTF2, but not PfGSTF58, became resistant to quizalofop-p-ethyl and haloxyfop-R-methyl. This reflects similar cross-resistance pattern to what was observed in the resistant P. fugax population. Transgenic rice seedlings overexpressing PfGSTF2 also exhibited resistance to quizalofop-p-ethyl. In contrast, CRISPR/Cas9 knockout of the orthologue gene in rice seedlings increased their sensitivity to quizalofop-p-ethyl. LC-MS analysis of in vitro herbicide metabolism by Escherichia coli-expressed recombinant PfGSTF2 revealed that quizalofop (but not haloxyfop) was detoxified at the ether bond, generating the GSH-quizalofop conjugate and a propanoic acid derivative with greatly reduced herbicidal activity. Equally, these two metabolites accumulated at higher levels in the resistant population than the susceptible population. In addition, both recombinant PfGSTF2 and PfGSTF58 can attenuate cytotoxicity by reactive oxygen species (ROS), suggesting a role in plant defence against ROS generated by herbicides. Furthermore, the GST inhibitor (NBD-Cl) reversed resistance in the resistant population, and PfGSTF2 (but not PfGSTF58) responded to NBD-Cl inhibition. All these suggest that PfGSTF2 plays a significant role in the evolution of quizalofop resistance through enhanced herbicide metabolism in P. fugax.

Enhanced Herbicide Metabolism and Target Site Mutation Enabled the Multiple Resistance to Cyhalofop-butyl, Florpyrauxifen-benzyl, and Penoxsulam in Echinochloa crus-galli.

This study investigated the multiple herbicide resistance (MHR) mechanism of one Echinochloa crus-galli population that was resistant to florpyrauxifen-benzyl (FPB), cyhalofop-butyl (CHB), and penoxsulam (PEX). This population carried an Ala-122-Asn mutation in the acetolactate synthase (ALS) gene but no mutation in acetyl-CoA carboxylase (ACCase) and transport inhibitor response1 (TIR1) genes. The metabolism rate of PEX was 2-fold higher, and the production of florpyrauxifen-acid and cyhalofop-acid was lower in the resistant population. Malathion and 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) could reverse the resistance, suggesting that cytochrome P450 (CYP450) and glutathione S-transferase (GST) contribute to the enhanced metabolism. According to RNA-seq and qRT-PCR validation, two CYP450 genes (CYP71C42 and CYP71D55), one GST gene (GSTT2), two glycosyltransferase genes (rhamnosyltransferase 1 and IAAGLU), and two ABC transporter genes (ABCG1 and ABCG25) were induced by CHB, FPB, and PEX in the resistant population. This study revealed that the target mutant and enhanced metabolism were involved in the MHR mechanism in E. crus-galli.

Transcriptomic analysis of maize uncovers putative genes involved in metabolic detoxification under four safeners treatment

Isoxadifen-ethyl (IDF) and cyprosulfamide (CSA) can effectively protect maize from nicosulfuron (NIC) injury, while mefenpyr-diethyl (MPR) and fenchlorazole-ethyl (FCO) did not. Their chemical diversity and requirement to use them in combination with the corresponding herbicides suggest that their elicitation of gene expression are complex and whether it is associated with the safening activity remains elusive. In this study, our first objective was to determine whether or not the ability of four safeners to enhance the metabolic rate of nicosulfuron. It was found that nicosulfuron degradation in maize was accelerated by IDF and CSA, but not by FCO and MPR. Transcriptomic analysis showed that the number of genes induced by IDF and CSA were larger than that induced by FCO and MPR. Overall, 34 genes associated with detoxification were identified, including glutathione S-transferase (GST), cytochrome P450 (CYP450), UDP-glucosyltransferase (UGT), transporter and serine. Moreover, 14 detoxification genes were screened for further verification by real-time PCR in two maize inbred lines. Two maize inbred lines exhibited high expression levels of four genes (GST31, GST39, AGXT2 and ADH) after IDF treatment. GST6, GST19, MATE, SCPL18 and UF3GT were specifically up-regulated in telerant maize inbred line under IDF and IDF + NIC treatments. Seven genes, namely GST31, GST6, GST19, UF3GT, MATE, ADH and SCPL18, are induced by IDF and CSA to play a vital role in regulating the detoxification process of NIC. Accordingly, the GST activity in maize was accelerated by IDF and CSA, but not by FCO and MPR. This result is consistent with transcriptome and metabolic data.These results indicate that the mitigation of NIC damage is associated with enhanced herbicide metabolism. IDF and CSA were more effective in protecting maize from NIC injury due to their ability to enhance the detoxification of specific types of herbicides, compared to FCO and MPR. The chemical specificity of four safeners is attributed to the up-regulated genes related to the detoxification pathway.

Enhanced Herbicide Metabolism and Target-Site Mutations Confer Multiple Resistance to Fomesafen and Nicosulfuron in Amaranthus retroflexus L.

Amaranthus retroflexus L. is a highly competitive broadleaf weed of corn-soybean rotation in northeastern China. In recent years, the herbicide(s) resistance evolution has been threatening its effective management in crop fields. One resistant A. retroflexus (HW-01) population that survived the protoporphyrinogen oxidase (PPO) inhibitor fomesafen and acetolactate synthase (ALS) inhibitor nicosulfuron applied at their field-recommended rate was collected from a soybean field in Wudalianchi City, Heilongjiang Province. This study aimed to investigate the resistance mechanisms of fomesafen and nicosulfuron and determine the resistance profile of HW-01 to other herbicides. Whole plant dose-response bioassays revealed that HW-01 had evolved resistance to fomesafen (50.7-fold) and nicosulfuron (5.2-fold). Gene sequencing showed that the HW-01 population has a mutation in PPX2 (Arg-128-Gly) and a rare mutation in ALS (Ala-205-Val, eight/twenty mutations/total plants). In vitro enzyme activity assays showed that ALS extracted from the HW-01 plants was less sensitive to nicosulfuron (3.2-fold) than ST-1 plants. Pre-treatment with the cytochrome P450 inhibitors malathion, piperonyl butoxide (PBO), 3-amino-1,2,4-triazole (amitrole), and the GSTs inhibitor 4-chloro-7-nitrobenzofurazan (NBD-Cl) significantly increased fomesafen and nicosulfuron sensitivity in the HW-01 population compared with that of the sensitive (S) population ST-1. Moreover, the rapid fomesafen and nicosulfuron metabolism in the HW-01 plants was also confirmed via HPLC-MS/MS analysis. Furthermore, the HW-01 population showed multiple resistance (MR) to PPO, ALS, and PSII inhibitors, with resistance index (RI) values ranging from 3.8 to 9.6. This study confirmed MR to PPO-, ALS-, and PSII-inhibiting herbicides in the A. retroflexus population HW-01, as well as confirming that the cytochrome P450- and GST-based herbicide metabolic along with TSR mechanisms contribute to their multiple resistance to fomesafen and nicosulfuron.

Target-site and non-target site resistance mechanisms are associated with iodosulfuron resistance in Lolium perenne L.

ABSTRACT Resistance to iodosulfuron, a herbicide targeting acetolactate synthase (ALS), was identified and characterised in a population of Lolium perenne L. from Canterbury, New Zealand. The resistant population was 264 times more resistant to iodosulfuron than a susceptible population, based on a ratio of the rate corresponding to a 50% reduction in resistant and susceptible plant survival (R/S LD50). The resistant population was also 14-times more resistant to nicosulfuron, another ALS-inhibitor of the same class. However, no resistance to imazapyr, an ALS-inhibitor from a different chemical class, was detected in the resistant population. Pre-treatment with an inhibitor of cytochrome P450 reduced the level of iodosulfuron resistance to 46-fold in the resistant population, suggesting enhanced herbicide metabolism, a non-target site mechanism, contributed to resistance of this herbicide. In addition, a proline to leucine substitution at codon 197 was detected, suggesting a target-site-based mechanism was also associated with resistance to iodosulfuron. This is the first report on the resistance mechanisms to ALS-inhibiting herbicides identified in Lolium perenne in New Zealand and highlights the importance of integrated weed management approaches to reduce the likelihood of ALS-inhibitor herbicide resistance becoming more widespread.

Fenclorim Increasing Butachlor Selectivity between Wheat and Roegneria kamoji by Seed Soaking

Roegneria kamoji Ohwi (Poaceae), a wild relative plant of wheat which is widely distributed across China, has become a dominant and problematic weed in wheat fields in some regions. We have previously confirmed that R. kamoji is highly tolerant to foliar-applied acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors in wheat (Triticum aestivum L.). The sensitivity of R. kamoji to pre-emergence (PRE) herbicides and the basis of fenclorim increase selectivity to butachlor between wheat and R. kamoji were evaluated in this study. Screenhouse bioassay showed that R. kamoji exhibited similar sensitivity to wheat to PRE herbicides at their recommended field doses (RFD); it also showed that buatchlor provides the highest relative control for R. kamoji (53.4% emergence and 81.5% fresh weight reduction, respectively), while it had no impact on seedling emergence of wheat among the six PRE herbicides. When butachlor was applied at four-fold RFD, no R. kamoji seedlings emerged; however, it significantly reduced the above-ground biomass of wheat compared with the non-treated control. Pre-treatment with herbicide safener fenclorim by seed soaking increased the ED10 value of butachlor to wheat from 221.8 to 1600.1 g a.i. ha−1, thus increasing the selectivity index from 9.6 to 68.9 between wheat and R. kamoji. The activities of α-amylase activity and protein content during germination, and glutathione-S-transferase (GST) and β-ketoacyl-CoA synthase (KCS) in the seedlings, could be induced by butachlor in both wheat seeds with or without fenclorim pre-soaking. These results suggested that butachlor provides the highest control for R. kamoji and did not affect germination and emergence in wheat. The basis of fenclorim-increased selectivity to butachlor was associated with the induced GST and KCS-mediated enhanced herbicide metabolism in wheat.

Investigation of resistance mechanisms to bentazone in multiple resistant Amaranthus retroflexus populations

Redroot amaranth (Amaranthus retroflexus L.) is a noxious weed that affects soybean production in China. Experiments were conducted to determine the molecular basis of resistance to bentazone. Whole-plant dose-response experiments showed that two populations (R1 and R2) exhibited resistance to bentazone with resistance indices of 9.01 and 6.85, respectively. Sequencing of the psbA gene revealed no amino acid substitution in the two populations. qRT–PCR analysis verified that psbA gene expression in R1 and R2 populations was increased significantly after treatment with bentazone, which was 3-fold and 5-fold higher than that in S1 and S2 populations, respectively. The P450 inhibitor malathion significantly reduced the level of resistance in the R1 and R2 populations when used prior to bentazone treatment. The R1 population exhibited multiple resistance to thifensulfuron-methyl and lactofen, caused by target site mutations (Asp-376-Glu in ALS, Arg-128-Gly in PPO2). In conclusion, increased gene expression of the psbA gene and enhanced herbicide metabolism seem to be the basis of resistance to bentazone in these A. retroflexus populations.

Comparison between the mechanisms of Clearfield ® wheat and Lolium rigidum multiple resistant to acetyl CoA carboxylase and acetolactate synthase inhibitors

Clearfield® wheat (Triticum aestivum) have helped eliminate the toughest grasses and broadleaf weeds in Spain since 2005. This crop production system includes other tolerant cultivars to the application of imidazolinone (IMI) herbicides. However, the continuous use and off-label rates of IMI herbicides can contribute to the development of resistance in Lolium rigidum and other weed species. In this research, the main objectives were to study the resistance mechanisms to acetolactate synthase (ALS) and acetyl coenzyme A carboxylase (ACCase) inhibitors in a L. rigidum accession (LrR) from a Clearfield® wheat field, with a long history rotating these IMI-tolerant crops and compare them with those present in the IMI-tolerant wheat. The resistance to ACCase inhibitors in LrR was due to point mutations (Ile1781Leu plus Asp2078Gly) of the target site gene plus an enhanced herbicide metabolism (EHM), on the other hand, in wheat accessions was due only by EHM. Mechanisms involved in the resistance to ALS inhibitors were both point mutations of the target gene and EHM in the IMI-tolerant wheat, while only evidence of mutation (Trp574Leu) was found in the multiple herbicide resistant L. rigidum accession. This research demonstrates that if crop rotation is not accompanied by the use of alternative sites of action in herbicide-tolerant crops, resistant weeds to herbicide to which crops are tolerant, can easily be selected. Moreover, repeated and inappropriate use of Clearfield® crops and herbicide rotations can lead to the evolution of multiple resistant weeds, as shown in this study, and have also inestimable environmental impacts.

Expression Pattern of Entire Cytochrome P450 Genes and Response of Defenses in a Metabolic-Herbicide-Resistant Biotype of Polypogon fugax.

Enhanced herbicide metabolism mediated by cytochrome P450s has been proposed as one of the major mechanisms of resistance to fenoxaprop-P-ethyl in a metabolic-herbicide-resistant biotype of Asia minor bluegrass (Polypogon fugax Nees ex Steud.). Upon pre-treatment with the P450 inhibitor piperonyl butoxide, a remarkable reduction in metabolic rates of the phytotoxic fenoxaprop-P has been observed in the resistant plants, implying that constitutive and/or fenoxaprop-P-ethyl-induced up-regulation of specific P450 isoforms are involved in the fenoxaprop-P-ethyl resistance. However, which P450 gene(s) were responsible for the metabolic resistance is still unknown. In this present study, based on the abundant gene resources of P. fugax established previously, a total of 48 putative P450 genes were isolated from the metabolic-herbicide-resistant plants and used for gene expression analysis. The most suitable reference genes for accurate normalization of real-time quantitative PCR data were first identified in P. fugax and recognized as actin (ACT), 18S rRNA (18S), and ribulose-1,5-bisphosphate carboxylase oxygenase (RUBP) under fenoxaprop-P-ethyl stress, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor 1α (EF1α) under mesosulfuron-methyl stress, and ACT, EF1α, eukaryotic initiation factor 4a (EIF4A), and 25S rRNA (25S) at different growth stages. Expression analysis of the putative P450 genes revealed that six genes, respectively, annotated as CYP709B1, CYP71A1-4, CYP711A1, CYP78A9, P450-11, and P450-39 were up-regulated more than 10-fold in the resistant plants by fenoxaprop-P-ethyl treatment, and all of them exhibited constitutively and/or herbicide-induced higher transcript levels in the fenoxaprop-P-ethyl-resistant than in the susceptible plants. Three genes, respectively, annotated as CYPRO4, CYP313A4, and CYP51H11 constantly up-regulated in the resistant than in the susceptible plants after fenoxaprop-P-ethyl treatment. Up-regulated expressions of these specific P450 genes were consistent with the higher P450 contents determined in the resistant plants. These results will help to elucidate the mechanisms for P450-mediated metabolic-herbicide resistance in P. fugax as well as other grass weed species.

The Basis of Tolerance Mechanism to Metsulfuron-Methyl in Roegneria kamoji (Triticeae: Poaceae).

Roegneria kamoji, a perennial monocot weed that belongs to the tribe Triticeae (family: Poaceae), is an emerging problematic weed in winter wheat (Triticum aestivum) fields in China. We have previously confirmed four R. kamoji populations tolerant to acetyl-CoA carboxylase (ACCase) inhibitors, and failed control of these populations by metsulfuron-methyl was observed. The objective of this study was to characterize the level of tolerance to metsulfuron-methyl, the basis of tolerance mechanism, and cross-tolerance to acetolactate synthase (ALS) inhibitors in R. kamoji. A whole-plant dose–response assay showed that plants of all R. kamoji populations (both from wheat fields and uncultivated areas) exhibited high tolerance to metsulfuron-methyl, based on their 100% survival at 6-fold recommended field dose (RFD) and ED50 values >6.84-fold RFD, no susceptible population was found. Gene sequencing indicated that no reported amino acid substitutions associated with resistance to ALS inhibitor were found in the ALS gene among the R. kamoji populations. Pretreatment with the known cytochrome P450 monooxygenases (CytP450) inhibitor malathion reduced the ED50 values of metsulfuron-methyl in two R. kamoji populations. These populations also exhibited cross-tolerance to RFD of mesosulfuron-methyl and bispyribac-sodium. The activities of glutathione-S-transferase (GST) and CytP450 could be induced by metsulfuron-methyl in R. kamoji, which is similar to the known tolerant crop wheat. This is the first report elucidating metsulfuron-methyl tolerance in R. kamoji. The reversal of tolerance by malathion and the GST and/or CytP450 enhanced herbicide metabolism suggests that non-target-site mechanisms confer tolerance to metsulfuron-methyl in R. kamoji.

Resistance Mechanism to Metsulfuron-Methyl in Polypogon fugax.

Polypogon fugax is a common winter weed in China and other Asia countries. We have previously found a P. fugax biotype (R) resistant to acetyl co-enzyme A carboxylase (ACCase) herbicides also cannot be effectively controlled by some acetolactate synthase (ALS) herbicides. This study evaluated the level of resistance to four ALS herbicides (metsulfuron-methyl, chlorsulfuron, monosulfuron, pyribambenz isopropyl) in the R biotype and the associated resistance mechanism. The R biotype exhibited moderate level of resistance to metsulfuron-methyl (6.0-fold) compared with the sensitive biotype (S). Sequence analysis of ALS gene revealed that two ALS genes existed in P. fugax. However, no substitution associated with ALS resistance mechanism were found in ALS genes between the S and R biotypes. The activity of ALS enzyme isolated from the R biotype was inherently higher and less sensitive to metsulfuron-methyl than the S biotype. Glutathione S-transferases (GST) activity was also less sensitive to metsulfuron-methyl in the R than as the S biotypes. Malathion, a cytochrome P450 (CYP) monooxygenase inhibitor, had much greater synergistic effect with metsulfuron-methyl on the R than as the S plants, reducing the ED50 value (herbicide dose to inhibit growth by 50%) of metsulfuron-methyl by 23- and 6-fold, respectively, suggesting that CYP mediated enhanced metabolism might contribute to the resistance to ALS herbicides. These results suggest that metsulfuron-methyl resistance in the R biotype was associated with the up-regulated ALS enzymatic activity and the GST and CYP-mediated enhanced herbicide metabolism.

Dinitroaniline Herbicide Resistance and Mechanisms in Weeds.

Dinitroanilines are microtubule inhibitors, targeting tubulin proteins in plants and protists. Dinitroaniline herbicides, such as trifluralin, pendimethalin and oryzalin, have been used as pre-emergence herbicides for weed control for decades. With widespread resistance to post-emergence herbicides in weeds, the use of pre-emergence herbicides such as dinitroanilines has increased, in part, due to relatively slow evolution of resistance in weeds to these herbicides. Target-site resistance (TSR) to dinitroaniline herbicides due to point mutations in α-tubulin genes has been confirmed in a few weedy plant species (e.g., Eleusine indica, Setaria viridis, and recently in Lolium rigidum). Of particular interest is the resistance mutation Arg-243-Met identified from dinitroaniline-resistant L. rigidum that causes helical growth when plants are homozygous for the mutation. The recessive nature of the TSR, plus possible fitness cost for some resistance mutations, likely slows resistance evolution. Furthermore, non-target-site resistance (NTSR) to dinitroanilines has been rarely reported and only confirmed in Lolium rigidum due to enhanced herbicide metabolism (metabolic resistance). A cytochrome P450 gene (CYP81A10) has been recently identified in L. rigidum that confers resistance to trifluralin. Moreover, TSR and NTSR have been shown to co-exist in the same weedy species, population, and plant. The implication of knowledge and information on TSR and NTSR in management of dinitroaniline resistance is discussed.

Non-target Site Herbicide Resistance Is Conferred by Two Distinct Mechanisms in Black-Grass (Alopecurus myosuroides).

Non-target site resistance (NTSR) to herbicides in black-grass (Alopecurus myosuroides) results in enhanced tolerance to multiple chemistries and is widespread in Northern Europe. To help define the underpinning mechanisms of resistance, global transcriptome and biochemical analysis have been used to phenotype three NTSR black-grass populations. These comprised NTSR1 black-grass from the classic Peldon field population, which shows broad-ranging resistance to post-emergence herbicides; NTSR2 derived from herbicide-sensitive (HS) plants repeatedly selected for tolerance to pendimethalin; and NTSR3 selected from HS plants for resistance to fenoxaprop-P-ethyl. NTSR in weeds is commonly associated with enhanced herbicide metabolism catalyzed by glutathione transferases (GSTs) and cytochromes P450 (CYPs). As such, the NTSR populations were assessed for their ability to detoxify chlorotoluron, which is detoxified by CYPs and fenoxaprop-P-ethyl, which is acted on by GSTs. As compared with HS plants, enhanced metabolism toward both herbicides was determined in the NTSR1 and NTSR2 populations. In contrast, the NTSR3 plants showed no increased detoxification capacity, demonstrating that resistance in this population was not due to enhanced metabolism. All resistant populations showed increased levels of AmGSTF1, a protein functionally linked to NTSR and enhanced herbicide metabolism. Enhanced AmGSTF1 was associated with increased levels of the associated transcripts in the NTSR1 and NTSR2 plants, but not in NTSR3, suggestive of both pre- and post-transcriptional regulation. The related HS, NTSR2, and NTSR3 plants were subject to global transcriptome sequencing and weighted gene co-expression network analysis to identify modules of genes with coupled regulatory functions. In the NTSR2 plants, modules linked to detoxification were identified, with many similarities to the transcriptome of NTSR1 black-grass. Critical detoxification genes included members of the CYP81A family and tau and phi class GSTs. The NTSR2 transcriptome also showed network similarities to other (a)biotic stresses of plants and multidrug resistance in humans. In contrast, completely different gene networks were activated in the NTSR3 plants, showing similarity to the responses to cold, osmotic shock and fungal infection determined in cereals. Our results demonstrate that NTSR in black-grass can arise from at least two distinct mechanisms, each involving complex changes in gene regulatory networks.

Loss of trifluralin metabolic resistance in Lolium rigidum plants exposed to prosulfocarb recurrent selection.

Resistance to the dinitroaniline herbicide trifluralin in Lolium rigidum (annual ryegrass) often is mediated by the enhanced capacity to metabolize the herbicide to less toxic polar conjugates and/or by functionally recessive target-site mutations in α-tubulin. In two L. rigidum populations possessing enhanced trifluralin metabolism, resistance was largely reversed by recurrent selection with the thiocarbamate herbicide prosulfocarb (i.e. plant survival was two- to >20-fold lower). Their ability to metabolize trifluralin was significantly decreased (by ≈2.3-fold) following recurrent prosulfocarb selection, to levels comparable to those observed in susceptible plants or when trifluralin metabolism was inhibited by treatment with the insecticide phorate. This study provides evidence that trait(s) enabling efficient trifluralin metabolism in L. rigidum are purged from the population under prosulfocarb recurrent selection. The level of trifluralin metabolism in vitro and its inhibition caused by phorate action on trifluralin-metabolizing enzyme(s) is equivalent to the effect produced by prosulfocarb selection. The hypothetical link between the two phenomena is that the putative monooxygenase(s) conferring trifluralin metabolic resistance also mediate the activation of prosulfocarb to its toxic sulfoxide. Thus, we speculate that survival to prosulfocarb via a lack of metabolic herbicide activation, and survival to trifluralin conferred by enhanced herbicide metabolism, are mutually exclusive. These findings not only open up a new research direction in terms of the interaction between different herbicide resistance mechanisms in L. rigidum, but also offer strategies for immediate management of the population dynamics of metabolism-based resistance in the field. © 2020 Society of Chemical Industry.

Metabolic resistance to S-metolachlor in two waterhemp (Amaranthus tuberculatus) populations from Illinois, USA.

Two waterhemp (Amaranthus tuberculatus) populations from Illinois demonstrating multiple-resistance to acetolactate synthase (ALS)-, 4-hydroxyphenylpyruvate dioxygenase, and photosystem II (PSII)-inhibiting herbicides (designated CHR and SIR) also displayed reduced sensitivity to very-long-chain fatty acid-inhibiting herbicides, including S-metolachlor. We hypothesized that a physiological mechanism, such as enhanced metabolism, could be responsible for the reduced efficacy of S-metolachlor. Metabolism experiments indicated that resistant populations degraded S-metolachlor more rapidly than sensitive populations and equally as rapidly as corn 2-24 h after treatment (HAT). Resistant waterhemp and corn metabolized 90% (DT90 ) of absorbed S-metolachlor in less than 3.2 h whereas DT90 values for sensitive waterhemp exceeded 6 h. The glutathione S-transferase inhibitor 4-chloro-7-nitrobenzofurazon and cytochrome P450-inhibitor malathion decreased the amount of S-metolachlor metabolized in resistant waterhemp at 4 HAT but not in sensitive waterhemp or corn, and altered the abundance of certain metabolites in resistant waterhemp. Results from this research demonstrate that resistance to S-metolachlor in these waterhemp populations is due to enhanced herbicide metabolism relative to sensitive populations. In addition, our results indicate that resistant waterhemp might utilize metabolic pathway(s) more intricate than either sensitive waterhemp or corn based on their metabolite profiles. © 2020 Society of Chemical Industry.

Safening activity and metabolism of the safener cyprosulfamide in maize and wheat.

Safeners extend the application of existing herbicides by selectively enhancing tolerance in large-grained cereal crops. While their activity is linked to enhanced herbicide metabolism, their exact mode of action and reasons for their crop specificity have yet to be determined. In this study, we investigated the selectivity of the recently developed sulfonamide safener cyprosulfamide (CSA) in maize (Zea mays L.) and wheat (Triticum aestivum), focusing on its uptake, distribution and metabolism in the two species. CSA protected maize, but not wheat, from injury by thiencarbazone-methyl (TCM). This correlated with the selective enhanced detoxification of the herbicide in maize. CSA underwent more rapid metabolism in maize than in wheat, with the formation of a specific hydroxylated metabolite correlating with safening. Studies with the nsf1 mutant sweetcorn line showed that the hydroxylation of CSA was partly mediated by the cytochrome P450 CYP81A9. However, primary metabolites of CSA were chemically synthesised and tested for their ability to safen TCM in maize but when tested were inactive as safeners. The results of this study suggest that the protection against TCM injury by CSA is linked to enhanced herbicide metabolism. This selective activity is due to the specific recognition of parent CSA in maize but not in wheat. Subsequent rapid oxidative metabolism of CSA led to its inactivation, demonstrating that cytochrome P450s regulate the activity of safeners as well as herbicides. © 2020 Society of Chemical Industry.

Non-target-site resistance to PDS-inhibiting herbicides in a wild radish (Raphanus raphanistrum) population.

Diflufenican resistance has been reported in wild radish populations since 1998, but the resistance mechanisms have not been investigated. Recently, we identified a wild radish population (H2/10) from the Western Australian grain belt that is resistant (R) to the phytoene desaturase (PDS)-inhibiting herbicide diflufenican. Dose-response results showed this R population is 4.9-fold more resistant than the susceptible (S) population based on the LD50 R/S ratio. In addition, the R population also exhibits cross-resistance to the PDS-inhibiting herbicide fluridone. The cytochrome P450 inhibitor malathion reversed diflufenican resistance and partially reversed fluridone resistance in the R population. The full coding sequences of the PDS gene were cloned from the S and R plants and there are natural variations in the PDS gene transcripts/alleles with no correlation to resistance. In addition, the R plants had a level of PDS gene expression that is not significantly different from the S plants. These results demonstrated that diflufenican resistance in this R wild radish population is likely due to non-target-site based enhanced herbicide metabolism involving cytochrome P450s. © 2019 Society of Chemical Industry.

Evolution of resistance to HPPD-inhibiting herbicides in a wild radish population via enhanced herbicide metabolism.

Relatively new herbicides that target 4-hydroxyphenylpyruvate dioxygenase (HPPD) are now available for use on the world's great grain crops (rice, wheat, corn and soybean) and for other uses. With widespread and persistent use of HPPD-inhibiting herbicides, the evolution of HPPD-inhibiting herbicide resistant weeds is inevitable. Currently, resistance to HPPD-inhibiting herbicides is known in two weed species, waterhemp and Palmer amaranth. Here, we report a HPPD-inhibiting herbicide resistant wild radish population from the Western Australia grain belt. This population was not selected with HPPD-inhibiting herbicides, rather it evolved resistance to earlier used herbicides with different modes of action and exhibits cross-resistance to HPPD-inhibiting herbicides. Dose-response experiments showed the resistant (R) population exhibits 4 to 6.5-fold resistance to the HPPD-inhibiting herbicides mesotrione, tembotrione and isoxaflutole, compared to the susceptible (S) population. This resistance is not target-site based as cloning of full coding sequences of the HPPD genes from S and R plants did not reveal resistance-endowing single nucleotide polymorphisms. The HPPD gene expression levels are similar in S and R plants. In addition, no differences in [14 C]-mesotrione uptake and translocation were observed in the S and R plants. However, the time required for R plants to metabolise 50% [14 C]-mesotrione is 7.7-fold faster than for the S plants. We confirm resistance to HPPD-inhibiting herbicides exists in a population of the economically damaging global weed wild radish. The resistance in this population is due to a non-target-site based enhanced rate of herbicide metabolism. © 2019 Society of Chemical Industry.

The First Case of Short-Spiked Canarygrass (Phalaris brachystachys) with Cross-Resistance to ACCase-Inhibiting Herbicides in Iran

The weed Phalaris brachystachys Link. severely affects winter cereal production. Acetyle-CoA Carboxylase (ACCase)-inhibiting herbicides are commonly used to control this weed in wheat fields. Thirty-six populations with suspected resistance to ACCase-inhibiting herbicides were collected from wheat fields in the Golestan Province in Iran. A rapid test performed in Petri dishes and whole-plant dose–response experiments were conducted to confirm and investigate the resistance level of P. brachystachys to ACCase-inhibiting herbicides. The seed bioassay results showed that 0.02 mg ai L−1 clodinafop-propargyl (CP) and 1.36 mg ai L−1 of the diclofop-methyl (DM) solution were the optimal amounts for reliably screening resistant and susceptible P. brachystachys populations. In the whole plant bioassay, all populations were found to be resistant to CP, resistance ratios ranging from 2.7 to 11.6, and all of the CP-resistant populations exhibited resistance to DM. Fourteen populations showed low resistance to cycloxydim, and thirteen of these populations were also 2-fold resistant to pinoxaden. The results showed that DM resistance in some P. brachystachys populations is likely due to their enhanced herbicide metabolism, which involves Cytochrome P450 monooxygenases, as demonstrated by the indirect assay. This is the first report confirming the cross-resistance of ACCase-inhibiting herbicides in P. brachystachys in Iran.

Alterations in Life-History Associated With Non-target-site Herbicide Resistance in Alopecurus myosuroides.

The evolution of resistance to herbicides is a classic example of rapid contemporary adaptation in the face of a novel environmental stress. Evolutionary theory predicts that selection for resistance will be accompanied by fitness trade-offs in environments where the stress is absent. Alopecurus myosuroides, an autumn-germinating grass weed of cereal crops in North-West Europe, has evolved resistance to seven herbicide modes-of-action, making this an ideal species to examine the presence and magnitudes of such fitness costs. Here, we use two contrasting A. myosuroides phenotypes derived from a common genetic background, one with enhanced metabolism resistance to a commercial formulation of the sulfonylurea (ALS) actives mesosulfuron and iodosulfuron, and the other with susceptibility to these actives (S). Comparisons of plant establishment, growth, and reproductive potential were made under conditions of intraspecific competition, interspecific competition with wheat, and over a gradient of nitrogen deprivation. Herbicide dose response assays confirmed that the two lines had contrasting resistance phenotypes, with a 20-fold difference in resistance between them. Pleiotropic effects of resistance were observed during plant development, with R plants having a greater intraspecific competitive effect and longer tiller lengths than S plants during vegetative growth, but with S plants allocating proportionally more biomass to reproductive tissues during flowering. Direct evidence of a reproductive cost of resistance was evident in the nitrogen deprivation experiment with R plants producing 27% fewer seed heads per plant, and a corresponding 23% reduction in total seed head length. However, these direct effects of resistance on fecundity were not consistent across experiments. Our results demonstrate that a resistance phenotype based on enhanced herbicide metabolism has pleiotropic impacts on plant growth, development and resource partitioning but does not support the hypothesis that resistance is associated with a consistent reproductive fitness cost in this species. Given the continued difficulties associated with unequivocally detecting costs of herbicide resistance, we advocate future studies that adopt classical evolutionary quantitative genetics approaches to determine genetic correlations between resistance and fitness-related plant life history traits.